Treatment of tobacco material

ABSTRACT

A method is provided for treating a tobacco material, wherein the method comprises treating the tobacco material with subcritical water. Also provided is a tobacco material which has been treated by such a method, or a derivative thereof, and a smoking article which comprises a tobacco material treated by such a method.

FIELD OF THE INVENTION

The present invention relates to a method for the treatment of tobaccomaterial.

BACKGROUND

In some circumstances, it may be desirable to reduce the content ofcertain constituents from tobacco material before incorporating thetobacco material into a smoking article, such as a cigarette. Forexample, it may be desirable to reduce the protein content of tobaccomaterial. Methods attempting to remove proteins have been proposed,although they have tended to be expensive, lengthy, and/or detrimentalto the physical structure of the tobacco material, and/or not reduce theprotein content to a desired level.

SUMMARY

According to a first aspect, there is provided a method for treating atobacco material, wherein the method comprises treating the tobaccomaterial with subcritical water.

According to a second aspect, there is provided a tobacco material whichhas been treated by a method according to the first aspect, or aderivative thereof.

According to a third aspect, there is provided a smoking articlecomprising a tobacco material according to the second aspect.

According to a fourth aspect of the present invention, there is providedthe use of subcritical water for removing one or more polyphenols orproteins from a tobacco material.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings, in which:

FIG. 1 shows a schematic of the Dionex ASE 350 pressurised hot waterextraction equipment.

FIGS. 2 and 3 show the physical properties of tobacco material treatedin accordance with a method of the invention with a static time of 30minutes.

FIG. 4 shows the physical properties of tobacco material treated inaccordance with the invention at different static times.

FIG. 5 shows an HPLC trace of an untreated tobacco material.

FIG. 6 shows the concentration of polyphenols in a tobacco materialtreated in

accordance with the invention with a static time of 45 minutes.

FIG. 7 shows concentration of protein in a tobacco material treated inaccordance with the invention.

FIG. 8 is a schematic side view of a smoking article including treatedtobacco material according to embodiments of the invention.

FIG. 9 shows the chemical structure of the four reference polyphenolcompounds detected and measured in experiments using HPLC: scopoletin,caffeic acid, chlorogenic acid, and rutin.

DETAILED DESCRIPTION

There is provided a method for treating a tobacco material, wherein themethod comprises treating the tobacco material with subcritical water.Subcritical water is liquid water under pressure at a temperaturebetween its conventional boiling point and its critical temperature,i.e. between 100° C. and 374° C. Subcritical water may also be referredto as “superheated water” or “pressurized hot water”.

Treating the tobacco material with subcritical water may be used for thepurpose of modifying the tobacco material in any suitable way. In someembodiments, treatment with subcritical water leads to the removal ofone or more chemical substances. In particular, in some embodiments,treatment with subcritical water leads to the removal of one or moreundesirable substances. In some embodiments, treatment with subcriticalwater leads to the removal of one or more polyphenols. In someembodiments, the treatment with subcritical water leads to the removalof one or more polyphenols selected from the group consisting ofchlorogenic acid, caffeic acid, scopoletin, quercetin, and rutin. Inparticular, in some embodiments the treatment with subcritical waterleads to the removal of chlorogenic acid and/or rutin. In someembodiments, the treatment with subcritical water may lead to theremoval of one or more proteins. In some embodiments, the treatment withsubcritical water may lead to the removal of one or more polyphenols andone or more proteins.

In some embodiments, treatment of the tobacco material with subcriticalwater results in a reduction in the content of one or more polyphenolsof at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 6o%, 70%, 75%,80%, 85%, 90%, 95% or a reduction in the content of one or morepolyphenols of 100%, based upon the polyphenol content of the untreatedtobacco material.

In some embodiments, the treatment of the tobacco material withsubcritical water results in the extraction of one or more polyphenolsin an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, based upon the polyphenolcontent of the untreated tobacco material.

Alternatively or in addition, the treatment with subcritical waterresults in a reduction in the protein content of at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or areduction in the protein content of 100%, based upon the protein contentof the untreated tobacco material.

In some embodiments, the treatment of the tobacco material withsubcritical water results in the extraction of protein in an amount ofat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90% or at least 95% or 100%, based upon the protein content of theuntreated tobacco material.

The method of the invention comprises at least one step in which thetobacco material is treated with subcritical water (“subcritical watertreatment step”). In some embodiments, where the method comprises morethan one subcritical water treatment step, the same or differentconditions may be employed in each subcritical water treatment step.

The subcritical water treatment step employed in the method of thepresent invention may involve contacting the tobacco material withsubcritical water. In some embodiments, the method may involvesubmerging the tobacco material in subcritical water. In someembodiments, the method involves submerging the tobacco material inwater at ambient temperature and subsequently increasing the pressureand temperature. This increase in pressure and temperature providessubcritical water in which the tobacco material is submerged. Forexample, the method may involve first increasing the pressure and thenincreasing the temperature of the water so as to provide the tobaccomaterial submerged in subcritical water.

Subcritical water exists under pressure, i.e. at an increased pressurein comparison to atmospheric pressure. The pressure employed in themethod of the present invention maybe any pressure suitable forproviding subcritical water. That is, a pressure at which liquid waterexists at a temperature between 100° C. and 374° C. In some embodiments,the method of the present invention comprises treating the tobaccomaterial with subcritical water at a pressure of from about 1500 psi toabout 1700 psi (from about 100 bar to about 120 bar). In someembodiments, the pressure is about 1500 psi (about 100 bar).

Subcritical water exists at temperatures between 100° C. and 374° C. Thetemperature employed in the method of the present invention may be atemperature that provides liquid water at a temperature between 100° C.and 374° C. In some embodiments, the treatment step is carried out at atemperature of at least 125° C. In some embodiments, the treatment stepis carried out at a temperature between about 100° C. and about 220° C.or between about 120° C. and about 200° C. or between about 125° C. andabout 175° C. In some embodiments, the treatment step is carried out ata temperature between about 125° C. and about 150° C.

In some embodiments, the water employed in the method may bede-oxygenated water. For example, the water may be degassed using asonicated bath to remove dissolved oxygen. In some embodiments, thewater employed is HPLC grade water.

In some embodiments, treating the tobacco material with subcriticalwater is a static treatment. For example, the tobacco material may besubmerged in subcritical water for a period of time; referred to as the“static period”.

The static period may be any length of time that allows the tobaccomaterial to be modified in the required way. In some embodiments, themethod of the present invention involves a static period of up to 2hours. In some embodiments, the method involves a static period of up to1 hour. In some embodiments, the method involves a static period ofbetween about 5 minutes and about 55 minutes or between about 15 minutesand about 45 minutes.

After treatment with subcritical water, the tobacco material may beseparated from the water (also referred to as the liquid extract). Thisseparation may involve any suitable filtration method, any suitablefiltering medium pore size, and any suitable number of filtration steps.For example, the tobacco material may be filtered by paper filtration,nanofiltration, microfiltration, and/or ultrafiltration. Alternativelyor in addition, the tobacco material may be separated from the liquidextract by centrifugation using any suitable centrifuge system, anysuitable angular velocity, and any suitable number of centrifugationsteps.

In some embodiments, the methods of the present invention thereforefurther comprise the step of separating the tobacco material from thewater. The pressure at which the step of separating (whether byfiltration or by any other means) is carried out is independent of thepressure employed in the subcritical water treatment step. In someembodiments, the step of separating (whether by filtration or by anyother means) is carried out at the same pressure as the subcriticalwater treatment step.

The method of the present invention may involve one or more subcriticalwater treatment steps. In some embodiments, the method comprises two ormore (multiple) subcritical water treatment steps.

In some embodiments involving a static process, the methods of thepresent invention may involve one or more static subcritical watertreatment steps. In some embodiments involving a static process, themethod comprises two or more (multiple) static subcritical watertreatment steps. For example, the method of the present invention maycomprise: a first subcritical water treatment step comprising treating atobacco material with subcritical water by submerging it in subcriticalwater; a first subsequent separation step comprising separating thetobacco material from the subcritical water (e.g. by filtration); asecond subcritical water treatment step comprising treating a tobaccomaterial with subcritical water by submerging it in subcritical water;and a second subsequent separation step comprising separating thetobacco material from the subcritical water (e.g. by filtration). Insome embodiments, such a method of the present invention furthercomprises a third subcritical water treatment step comprising treating atobacco material with subcritical water by submerging it in subcriticalwater; and a third subsequent separation step comprising separating thetobacco material from the subcritical water (e.g. by filtration).

In some embodiments of the above multiple treatment/separationembodiments of the invention, each treatment step and each separationstep is carried out at the same pressure, e.g. from about 1500 psi toabout 1700 psi (from about 100 bar to about 120 bar).

Once separated from the water/liquid extract, the tobacco material (alsoreferred to as tobacco residue) may be washed any suitable number oftimes using any suitable liquid or liquids, such as water. In someembodiments, the methods of the invention further comprise a washingstep comprising washing the treated tobacco material with water.

In some embodiments, the method of the present invention furthercomprises a step of drying the treated, separated tobacco material. Forexample, the tobacco material may be dried using a centrifuge and/or inan oven.

Tobacco material comprises dead plant cells, and dead plant cells havemany functional groups. In some embodiments, the functional groups arereactive towards water under conditions provided by the invention. As aresult, exposing tobacco material to water under favourable conditionsis likely to result in the breakdown of different cellular structures,and the consequent release of different chemical substances. Mostsignificantly, cellulose in the plant cell walls comprises O-glycosidicbonds, which may be broken under favourable conditions to cause the cellwall to rupture, and the cell membrane to rupture—without the cell wallto balance the positive pressure potential of the water (Ψ_(p)), andmany intracellular substances to escape.

The method of the invention may be applied to any suitable tobaccomaterial. The tobacco material maybe derived from any suitable part ofany suitable tobacco plant of the plant genus Nicotiana. The tobaccomaterial may then be treated in any suitable way, and may be cured usingany suitable method of curing, before being treated according to themethod of the invention. In some embodiments, however, the tobaccomaterial treated by the method of the invention has already been curedand may be cured cut rag and/or cured whole leaf tobacco. Examples oftobaccos which may be used in the method of the invention include, butare not limited to: Virginia, Burley, Maryland, Oriental, and Rustica.

In some embodiments, the method of the invention—in particular the stepof treating tobacco material with subcritical water—reduces or minimisesthe removal of at least some of the chemical substances whose removalwould be undesirable, which could be the case for a variety of differentreasons. One reason, for example, could be that the substance makes apositive contribution to the experience of smoking a smoking articlewhich contains the treated tobacco material.

Nicotine may be an example of such a substance, and for this reason insome embodiments it is undesirable to remove this molecule. In someembodiments, the method of the invention removes less than 50%, 40%,30%, 20%, 10%, or 5% of the nicotine from the tobacco material; infurther embodiments, the method of the invention removes less than 2%,1%, 0.5%, or 0.1% of nicotine from the tobacco material; and, in furtherembodiments still, the method of the invention removes essentially nonicotine from the tobacco material.

In embodiments wherein treating the tobacco material with subcriticalwater leads to the removal of one or more chemical substances from thetobacco material, one or more of these may be re-introduced into thematerial following treatment, and one or more of these may be substanceswhose removal would be undesirable, such as nicotine.

In addition to one or more subcritical water treatment steps, the methodof the invention may comprise one or more further treatment steps.Further treatment steps maybe particularly useful in the method of theinvention for the purpose of removing large quantities of protein. Thisis because treatment with subcritical water is likely to rupture theplant cell walls in the tobacco material, thereby providing easieraccess to the intracellular components of the plant cells and theproteins found therein. Suitable additional treatment steps include, butare not limited to: treating the tobacco material with one or moresuitable non-ionic liquids, such as water; treating the tobacco materialwith one or more enzymes, which may be enzymes which catalyse themodification of polyphenols or proteins, such as phenol-oxidising andproteolytic enzymes; treating the tobacco material with one or moresuitable surfactants, such as sodium dodecylsulfate (SDS), in anysuitable solvent; treating the tobacco material with one or moresuitable adsorbent materials, such as polyvinyl polypyrrolidone (PVPP),hydroxylapatite, bentonite, activated carbon or attapulgite, in anysuitable solvent if appropriate; and treating the tobacco material withone or more suitable non-aqueous liquids, such as ionic liquids.

Additionally or alternatively, the tobacco material subjected tosubcritical water treatment may be subsequently subjected to furtherextraction processes.

Having undergone any of the previously-described treatment steps inaccordance with the method of the invention, the tobacco material may bedried and further modified in any suitable way before being incorporatedinto a smoking article. For example, certain chemical substances may beadded to the tobacco material, such as flavourants where localregulations permit, and the tobacco material maybe cut and/or shreddedbefore being incorporated into a smoking article using any suitablemethod of incorporation.

As used herein, the term “smoking article” includes smokeable productssuch as cigarettes, cigars and cigarillos whether based on tobacco,tobacco derivatives, expanded tobacco, reconstituted tobacco or tobaccosubstitutes and also heat-not-burn products. The smoking article may beprovided with a filter for the gaseous flow drawn by the smoker.

As used herein, the terms “flavour” and “flavourant” refer to materialswhich, where local regulations permit, maybe used to create a desiredtaste or aroma in a product for adult consumers. They may includeextracts (e.g., licorice, hydrangea, Japanese white bark magnolia leaf,chamomile, fenugreek, clove, menthol, Japanese mint, aniseed, cinnamon,herb, wintergreen, cherry, berry, peach, apple, Drambuie, bourbon,scotch, whiskey, spearmint, peppermint, lavender, cardamon, celery,cascarilla, nutmeg, sandalwood, bergamot, geranium, honey essence, roseoil, vanilla, lemon oil, orange oil, cassia, caraway, cognac, jasmine,ylang-ylang, sage, fennel, piment, ginger, anise, coriander, coffee, ora mint oil from any species of the genus Mentha), flavour enhancers,bitterness receptor site blockers, sensorial receptor site activators orstimulators, sugars and/or sugar substitutes (e.g., sucralose,acesulfame potassium, aspartame, saccharine, cyclamates, lactose,sucrose, glucose, fructose, sorbitol, or mannitol), and other additivessuch as charcoal, chlorophyll, minerals, botanicals, or breathfreshening agents. They may be imitation, synthetic or naturalingredients or blends thereof. They may be in any suitable form, forexample, oil, liquid, or powder.

Referring to FIG. 8, for purpose of illustration and not limitation, asmoking article l according to an exemplary embodiment of the inventioncomprises a filter 2 and a cylindrical rod of smokeable material 3, suchas tobacco treated in accordance with the invention described herein,aligned with the filter 2 such that one end of the smokeable materialrod 3 abuts the end of the filter 2. The filter 2 is wrapped in a plugwrap (not shown) and the smokeable material rod 3 is joined to thefilter 2 by tipping paper (not shown) in a conventional manner.

FIG. 10 shows, for illustration only, a flow chart setting out the stepsinvolved in one embodiment of the invention. The steps shown in FIG. 10should not be viewed as limiting the disclosure of the presentapplication as a whole.

The methods of the invention may comprise any suitable steps, and anysuitable number of steps, in order to reduce the polyphenol and/orprotein content of the tobacco material. The method of the invention mayalso further modify the tobacco material in any suitable way, forexample by modifying the flavour it generates upon combustion, and/orremoving other types of chemical substances.

In some embodiments, the methods described herein may comprise one ormore further steps to modify the tobacco material in any suitable way.For example, the tobacco material maybe modified to provide it with oneor more characteristics desirable for a tobacco material. For example,where the treated tobacco material is to be incorporated into a smokingarticle such as a cigarette, the tobacco material may be treated inorder to modify the flavour it generates upon combustion, and/or may betreated in order to remove one or more of its chemical substances.

Experimental Work

A series of experiments were carried out in order to investigate how thetreatment of a tobacco material with subcritical water can affect theprotein and polyphenol content of the tobacco material. The disclosedexperimental work is not intended to limit the scope of the invention.

Procedure

The experiments were conducted using a commercially available Dionex ASE350 system. The Dionex uses a static extraction method and is capable ofheating water to a temperature of 200° C. while still remaining in aliquid state. Other systems can of course be used.

Merely for illustration, FIG. 1 shows a schematic of the Dionex ASE 350pressurised hot water extraction (PHWE) equipment, which is one type ofequipment that can be used to perform the method of the presentinvention. Of course, other equipment may also be used. Sample materialis packed into a 100 ml stainless steel sample cell fitted with a paperfilter at the base. The cell is then transferred to a preheated oven bya robotic arm. The cell and ASE equipment is capable of withstandingpressure of up to 3000 psi.

Once the sample is loaded into the pre heated oven a pump deliverssolvent at ambient temperature to the sample cell. The static releasevalve seals the cell automatically once solvent has travelled throughthe system to the collection vessel. The pump continues to pump solventuntil the pressure reaches 1500 psi. If the pressure reaches 1700 psi atany time during operation the static valve opens briefly to relieve thepressure. The pump also delivers fresh solvent to the cell to maintainhigh pressure. Once the cell is loaded the oven undergoes two heatingstages the first is an internally defined time for the cell and itscontents to reach thermal equilibrium with the oven, the second is astatic time where the cell is maintained at the required temperature.The static time is defined by the operator.

After the static period is complete the static valve is opened and thesolvent drained to the collection bottle. Fresh solvent is then pumpedthrough the cell to remove extracted materials. The amount of solventused here in this purging stage is defined by the operator. Finally thesolvent is purged out of the cell with nitrogen (150 psi). The cell isremoved from the oven by a mechanical arm and the residual pressurevented to atmosphere. With the cell removed from the circuit the entiresystem is purged with clean solvent to prevent contamination.

Materials and Methods

15 g of tobacco was loaded into a 100 ml ASE Cell, the cell was fittedwith a paper filter at the base. The water (HPLC grade purchased fromRathburn UK) was degassed for 25 mins using a sonicated bath to removedissolved oxygen that may cause oxidation at elevated temperature.

In the initial batch of experiments a total of eighteen 15 g sampleswere extracted under the following conditions.

TABLE 1 Extraction Conditions Mass of Tobacco (g) Temperature (° C.)Static Time (min) 15 75 15 15 75 30 15 75 45 15 100 15 15 100 30 15 10045 15 125 15 15 125 30 15 125 45 15 150 15 15 150 30 15 150 45 15 175 1515 175 30 15 175 45 15 200 15 15 200 30 15 200 45

In the second batch of extracts 2×15 g samples of tobacco were extractedat 125° C. and 150° C. The samples were heated for a total of 45 min,during this period the static valve was opened at 15 min intervals andthe cell drained of water. The water was collected and the cell was thenrefilled with fresh solvent.

After extraction the cells were allowed to cool briefly and theextracted fibre was dried using a centrifuge at 1000 rpm for 15 mins.The filtrate was combined with the primary water extract. The driedfibre was then dried in an oven at 75° C. for 12 hrs then at roomtemperature for 48 hrs.

The liquid extracts were transferred to weighed vessels, frozen thendried to a constant weight using a freeze drier. This took approximately3 days. After freeze drying the samples were weighed placed in airtightcontainers and stored at −4° C.

Fibre Sample Pre-Treatment

Prior to treatment in any of the following analytical techniques thefibre was ground to a fine powder using an in house modified smalldomestic blender. The material was continually ground until it wouldpass through a 40 mesh screen.

Identification and Quantification of Individual Phenolics via HighPerformance Liquid Chromatography (HPLC)

The dried tobacco residue and freeze-dried extracts were analysed usingHPLC. This was performed using an Agilent 1100 series HPLC fitted with aDiode array. Standards were supplied by Sigma Aldrich UK. HPLC solventswere supplied by Rathburn Chemicals UK.

Samples were diluted but shaken on an orbital shaker for 10 mins thensonicated using a sonic bath for a period of 20 mins. Following this thesamples were centrifuged using a desk centrifuge for a period of 15mins.

HPLC was used to measure the concentration of four reference polyphenolcompounds, namely scopoletin, caffeic acid, chlorogenic acid, rutin inthe aqueous filtrate following filtration. The chemical structures ofthese four reference polyphenol compounds are provided in FIG. 9.

Protein Nitrogen Determination

The dried tobacco residues were analysed using a combustion method. Theanalysis was performed using a LECO TruMac.

The amount of sample used was 1 g. Only the fibre could be analysed withthis technique as the extracts were highly soluble in water and washesto remove non-protein nitrogen were not possible.

While it is known that tobacco would contain non protein nitrogen fromalkaloids a pre-treatment with hot acetic acid washes is assumed to becapable of removing these.

Quantification of Total Phenolic Content—via Assay

Polyphenol content was determined for the freeze dried extracts and thetobacco residues using a Folin Ciocalteu (FC) method.

The FC assay is a colorimetric assay used to provide a measure of totalpolyphenol content in solution. In an FC assay, the magnitude ofabsorption at a particular radiation frequency—which polyphenolsabsorb—is measured for a sample. Following this, the measured magnitudeof absorption is compared to the magnitude of absorption at the sameradiation frequency for a solution of the polyphenol, Gallic Acid. Themeasured absorption of light may then be expressed in units of GAE(Gallic Acid Equivalents).

1 mg per ml samples of the freeze-dried extracts and a 10 mg per mlsamples of the dried fibre were prepared in a 0.14 M NaCl solution. Thesamples were shaken on an orbital shaker for 10 mins then sonicated fora period of 60 mins. Following this the samples were centrifuged using adesk centrifuge for a period of 15 mins. Following shaking and prior tocentrifugation the fibre samples were placed in an oven at 60° C. for 18hours to attempt to remove any phenolics that may still be bound to thecells of the fibre.

50 μl and 30 μl samples of the freeze-dried and fibre solutionsrespectively were added to 1 ml of 7% sodium carbonate in 5 ml testtubes, 100 μl of Folin Ciocalteu phenol reagent (Sigma UK) was added.The tubes were then stirred with a vortex mixer. The mixtures were thenincubated for 60 Mins at 40° C. The absorbance was measured at 688 nmusing a Pharmacia Novaspec 2 Spectrophotometer. Different concentrationsof gallic acid were used for the calibration curve.

Quantification of Total Protein Content Via Assay

Total protein content was determined for the freeze dried extracts andthe tobacco residues using a bichinchoninic acid assay.

1 mg per ml sample of the freeze-dried extracts and a 10 mg per mlsample of the dried fibre were prepared in a 0.14 M NaCl solution. Thesamples were shaken on an orbital shaker for 10 mins then sonicated fora period of 20 mins. Following this the samples were centrifuged for aperiod of 15 mins. Following shaking the fibre samples were placed in anoven at 60° C. for 18 hours to attempt to remove any protein that maystill be bound to the cells of the fibre, additionally 0.1%trifluoroacetic acid was added to the mixture to assist extraction ofthe protein.

30 μl samples of the freeze dried extract and fibre solutions were addedto 1 ml of solution containing Bichinchoninic acid solution and Copper(II) pentahydrate 4% mixed in a 50.1 ratio (Sigma UK). The mixtures werethen stirred with a vortex mixer and sealed with parafilm. The mixtureswere incubated for 20 mins at 40° C. The absorbance was measured at 562nm using a Novaspec 2 Spectrophotometer. Different concentrations ofBovine serum albumin (BSA) were used for the calibration curve.

Results & Discussion

Physical Properties of Extracts:

The masses of the dried tobacco residues are given in Table 2.

The temperatures 150 III and 125 III refer to the extractions wherethree extracts are produced from the same tobacco.

TABLE 2 Masses of Tobacco Residues Static time Mins Dried Fibre Mass g15 30 45 Temperature ° C.  75 7.52 7.52 7.61 100 7.55 7.18 7.23 125 6.746.07 6.14 150 6.11 4.91 4.65 175 5.05 4.62 4.91 200 3.76 4.12 4.14125III 5.5 150III 4.6

A clear trend is seen in the dried fibre mass, as the temperature isincreased the amount of material left as a fibre decreases.

As observed in FIGS. 2 and 3, the physical properties of the tobaccoresidue change dramatically as the temperature is increased. Up to 100°C. the material remains mostly unchanged and un-compacted. Between 125°C. and 150° C. the material begins to compact slightly althoughindividual strands/fibres can still be seen and broken up by crumbling,the material begins to take on a slightly darker colour. At 175° C. andabove the material begins to compact heavily, and the material takes ona dark brown black colour.

The effect of static time is not as significant as temperature on thematerial. FIG. 4 shows the material extracted at 125° C. for differentperiods. Some compacting and discolouration are observed, although theeffects are far less pronounced.

HPLC Quantification of Select Polyphenol

HPLC was employed to quantify polyphenols in the treated tobaccomaterial. An example trace of unextracted tobacco is shown in FIG. 5 forcomparison. The peak symmetry is good and the isomer of chlorogenic acid(cryptochlorogenic acid) is sufficiently separated from chlorogenic acidto allow accurate integration.

The HPLC traces for the extracts all show very similar trends. As thetemperature increases the amount of polyphenol in the fibre decreased.Plots of phenol content (FIG. 6) show the chlorogenic acid and rutindecrease at a very similar rate. The intensity of the caffeic acid peakis so low in virtually all traces that integration of the peak may notbe reliable.

Changing the static time does not greatly affect the amount ofpolyphenol in the samples.

For the experiments at 125° C. and 150° C. where the sample wasextracted three times no polyphenol is detected in the fibre. This isseen to be more effective than one extraction for 45 min.

The total amount of each polyphenol present in the start sample is shownin Table 3. To allow a direct comparison of this to the extracted fibrethe total amount of chlorogenic acid in the fibre is shown in Table 4.

TABLE 3 Amount of each selected polyphenol in the untreated tobaccototal mg of total mass of selected mg per G sample g polyphenolChlorogenic 12.97 15 194.6 acid Caffeic acid 0.14 15 2.1 Scopoletin 0.7115 10.6 Rutin 11.87 15 178.1

TABLE 4 Total amount of chlorogenic acid in the treated fibre for thematerial mg/g Total mg Temp ° C. Time Chlorogenic acid Total Mass gchlorogenic acid 75 15 3.006 7.52 22.607 100 15 3.115 7.55 23.519 125 152.244 6.74 15.126 150 15 3.605 6.11 22.029 175 15 1.252 5.05 6.323 20015 0.910 3.76 3.420 75 30 3.779 7.52 28.418 100 30 2.864 7.18 20.560 12530 1.823 6.07 11.064 150 30 1.140 4.91 5.598 175 30 1.137 4.62 5.254 20030 0.642 4.12 2.646 75 45 3.305 7.61 25.152 100 45 2.682 7.23 19.391 12545 1.734 6.14 10.649 150 45 1.138 5.2 5.920 175 45 0.861 4.91 4.227 20045 0.617 4.14 2.555 125 Triple 0.000 5.5 0.000 150 Triple 0.000 4.60.000

The table shows clearly that the total amount of chlorogenic acid in thefibre drops rapidly even when extracted with water at 75° C. for 15mins. The amount of chlorogenic acid decreases steadily as thetemperature is increased. The effect of time is seen to be lessinfluential. When the fibre was extracted with three volumes of freshwater no chlorogenic acid could be detected in the treated tobaccomaterial.

Full data for the other polyphenols is provided for the fibre in Table5. The trends shown by chlorogenic acid are mirrored by the otherpolyphenols. Some deviation was seen in results for scopoletin andcaffeic acid where the amount present in the fibre tends to fluctuate.This could be attributed to the low concentration being at the limits towhich the machine employed is accurate; some peaks were of such lowintensity manual integration was required.

TABLE 5 Total mg of selected phenol in fibre Total Amount of Polyphenolin extract mg Chlorogenic Caffeic Mass g Acid Acid Scopoletin RutinUnextracted 15 194.556 2.150 10.639 178.071 start material Mass of fibreChlorogenic Caffeic Temp Time extract Acid Acid Scopoletin Rutin ° C.mins g mg mg mg mg 75 15 7.52 22.61 0.61 1.85 27.55 100 15 7.55 23.520.37 1.38 30.31 125 15 6.74 15.13 0.00 0.82 18.39 150 15 6.11 22.03 0.232.60 22.53 175 15 5.05 6.32 0.13 1.43 5.24 200 15 3.76 3.42 0.00 1.171.52 75 30 7.52 28.42 0.41 1.71 36.02 100 30 7.18 20.56 0.00 1.06 24.52125 30 6.07 11.06 0.20 0.74 14.02 150 30 4.91 5.60 0.14 0.54 6.84 175 304.62 5.25 0.10 1.28 2.75 200 30 4.12 2.65 0.00 1.19 0.00 75 45 7.6125.15 0.59 1.74 32.57 100 45 7.23 19.39 0.42 1.14 22.99 125 45 6.1410.65 0.22 0.70 13.65 150 45 5.2 5.92 0.15 0.67 6.10 175 45 4.91 4.230.00 1.35 1.88 200 45 4.14 2.56 0.00 1.09 0.00 125 Triple 5.5 0.00 0.000.00 0.00 150 Triple 4.6 0.00 0.00 0.00 0.00

To show how the level of polyphenols is reduced in a typical treatmentstep, the polyphenol content of the untreated tobacco, and the treatedtobacco material at 75° C. for 15 mins are shown in Table 6.

TABLE 6 Phenolic content of untreated tobacco and tobacco treated at 75°C. for 15 mins Chlorogenic Caffeic acid Rutin acid Total ScopoletinTotal Total mass mass Total mass mass present present present presentmg/g mg mg/g mg mg/g mg mg/g mg Untreated 12.97 194.56 0.14 2.15 0.7110.64 11.87 178.07 tobacco Fibre 3.01 22.61 0.08 0.61 0.25 1.85 3.6627.55

Total Phenolic Content

The total phenolic content of the treated tobacco material as expressedin Gallic acid equivalence (GAE) is shown in Table 8 below. The datashows a significant reduction in total phenolic content compared to theuntreated tobacco.

When the fibres extracted three times at 125 and 150° C. were analysedvery low concentrations of phenolics were detected. These values alongwith the untreated tobacco are also shown in Table 7. The data showsclearly three extractions remove a far greater amount of phenolics fromthe tobacco.

TABLE 7 Total GAE of untreated tobacco and fibre GAE equivalent Mass ofmg/G sample g Total GAE mg in Fibre Start Material 33.8 15 507.1 Temptime  75-15 20.7 7.52 155.5 100-15 14.7 7.55 111.0 125-15 12.4 6.74 83.5150-15 19.7 6.11 120.5 175-15 19.3 5.05 97.6 200-15 20.5 3.76 77.0 75-30 28.4 7.52 213.6 100-30 15.3 7.18 109.7 125-30 8.5 6.07 51.8150-30 7.9 4.91 39.0 175-30 18.2 4.62 84.0 200-30 18.8 4.12 77.3  75-4528.8 7.61 219.0 100-45 18.0 7.23 130.0 125-45 8.3 6.14 51.2 150-45 14.75.2 76.4 175-45 16.1 4.91 78.8 200-45 26.3 4.14 108.8 125° C. Triple 3.15.5 17.2 extract 150° C. Triple 1.8 4.6 8.2 extract

Similar to the HPLC results for the selected polyphenols a largereduction in polyphenol is seen even at 75° C. for 15 mins. Increasingthe temperature to 125° C. causes the amount of polyphenol to dropfurther.

Protein Determination By Assay

As observed in FIG. 7, the amount of protein present in the fibrefollows a rapid descent from 75° C. to 125° C. after this point theamount of protein stabilises and increasing temperature has littleeffect. This could be due to the maximum amount of protein being removedat 125° C.

By comparison the amount of protein extracted with a static time of 45mins is shown in Table 8. For comparison the untreated tobacco isincluded. The concentration (mg/g) of protein in some of the extracts ishigher than that of the untreated material, although when the mass ofthe extract is take into account it is seen that the total amount ofprotein is less. Without being bound by theory, we attribute theseresults to low temperature water selectively removing non-proteinmaterials.

TABLE 8 Amount of protein present in fibre - static time 45 mins mgprotein present in mg/g protein Mass g sample Untreated 90.0 15.0 1350.0 75 130.2 7.6 990.4 100 99.3 7.2 717.8 125 76.9 6.1 471.9 150 79.9 5.2415.2 175 64.9 4.9 318.7 200 83.3 4.1 345.0

In order to address various issues and advance the art, the entirety ofthis disclosure shows by way of illustration various embodiments inwhich the claimed invention(s) maybe practiced and provide for superiortobacco treatment, tobacco material, and products incorporating tobaccomaterial. The advantages and features of the disclosure are of arepresentative sample of embodiments only, and are not exhaustive and/orexclusive. They are presented only to assist in understanding and teachthe claimed features. It is to be understood that advantages,embodiments, examples, functions, features, structures, and/or otheraspects of the disclosure are not to be considered limitations on thedisclosure as defined by the claims or limitations on equivalents to theclaims, and that other embodiments may be utilised and modifications maybe made without departing from the scope and/or spirit of thedisclosure. Various embodiments may suitably comprise, consist of, orconsist essentially of, various combinations of the disclosed elements,components, features, parts, steps, means, etc. In addition, thedisclosure includes other inventions not presently claimed, but whichmay be claimed in future.

1. A method for treating a tobacco material, the method comprising:receiving a tobacco material; and treating the tobacco material withsubcritical water at a pressure from about 10×10⁶ Pa (1500 psi) to about12×10⁶ Pa (1700 psi).
 2. The method according to claim 1, wherein thetreating includes removing one or more polyphenols from the tobaccomaterial.
 3. The method according to claim 1, wherein the treatingincludes removing one or more proteins from the tobacco material.
 4. Themethod according to claim 1, wherein the treating is carried out at atemperature from about 125° C. to about 175° C.
 5. The method accordingto claim 1, the method further comprising submerging the tobaccomaterial in subcritical water.
 6. The method according to claim 5,wherein treating the tobacco material with subcritical water is a statictreatment.
 7. The method according to claim 6, wherein the submerging isa first submerging, the method further comprising a first separating ofthe tobacco material from the subcritical water after the firstsubmerging.
 8. The method according to claim 7, the method furthercomprising: a second submerging of the tobacco material in subcriticalwater after the first separating; and a second separating of the tobaccomaterial from the subcritical water after the second submerging.
 9. Themethod according to claim 8, the method further comprising: a thirdsubmerging of the tobacco material in subcritical water after the secondseparating; and a third separating of the tobacco material from thesubcritical water after the third submerging.
 10. The method accordingto claim 1, tobacco material is separated from the subcritical water byfiltration.
 11. The method according to claim 1, the method furthercomprising drying the treated tobacco material.
 12. A tobacco materialthat has been treated by the method according to claim
 1. 13. A smokingarticle a comprising the tobacco material according to claim
 12. 14.(canceled)
 15. The method according to claim 8, wherein, in the firstseparating and in the second separating, the tobacco material isseparated from the subcritical water by filtration.
 16. The methodaccording to claim 9, wherein, in the first separating, in the secondseparating, and in the third separating, the tobacco material isseparated from the subcritical water by filtration.